The microtubule cytoskeleton is required for cell division and other essential cell processes. Spatiotemporal regulation of the microtubule cytoskeleton is accomplished in part by the action of microtubule organizing centers (MTOCs), the most prominent of which is the centrosome in animal cells. The organizing potential of the centrosome relies on the pericentriolar material (PCM), a dynamic protein matrix that recruits microtubule nucleating factors. Remarkably, the PCM forms a unique cellular compartment of discrete size and composition, all in the absence of a delimiting membrane. Although the key protein components of the PCM have been identified through a combination of genetic and biochemical studies, the manner by which they interact to form the PCM structure and recruit microtubules is not understood. To address this question, this proposal will exploit new technologies in an innovative, integrative approach towards the following aims: 1) create a spatial and temporal map of the critical protein-protein interactions in the PCM, 2) test sufficiency and hierarchy of assembly by relocalizing PCM formation in cells, and 3) reconstitute the PCM in vitro from purified components. Together, these aims will identify the protein-protein interactions sufficient for building the microtubule organizing center, furthering our understanding of this important structure. Defects in centrosome structure, size, and function are associated with many disease states including cancer, microcephaly, and the ciliopathies. PCM formation specifically is dysregulated in cancer cells, which often exhibit increased PCM size and enhanced microtubule nucleation capacity. In addition, increased microtubule nucleation induces oncogene-like cell invasion in culture (Godinho et al., 2014). Thus, this work has the potential to improve our understanding of human disease, specifically that of the cancer cell phenotype.